In the field of analyzing macromolecules, one-dimensional and two-dimensional gel electrophoresis have become standard tools for separating and visualizing macromolecules. One-dimensional gel electrophoresis is used to separate mixtures of macromolecules, such as proteins, into individual components according to differences in mass by electrophoresing in a polyacrylamide gel under denaturing conditions.
Two-dimensional gel electrophoresis involves isoelectric focusing to separate proteins electrophoretically on the basis of their relative acidic and basic residue content. Under the influence of an applied electric field, a more highly charged protein will move faster than a less highly charged protein of similar size and shape. If the proteins are made to move from a sample zone through a non-convecting medium (e.g., polyacrylamide), an electrophoretic separation will result. When the protein enters a region that has a pH value at which the protein's net charge is zero (the isoelectric point), it will cease to migrate relative to the medium. Further, if the migration occurs through a pH gradient that increases monotonically from the anode, the protein will “focus” at its isoelectric point.
Polyacrylamide gels comprising polyacrylamide cross-linked by bis-acrylamide are primarily the medium of choice for protein analysis. Currently available gels are non-dissolvable and repeatedly show retention of proteins after transfer to the second dimension. Compounding this problem, once resolved onto the second dimension, further protein retention and losses occur with preceding manipulations. Although yields approach 80-90% recovery using traditional methods (i.e., mechanical, electro-elution, and diffusion methods), total recovery is rarely achieved. Further, chemical disruption of the gel matrix band often modifies the protein, in turn altering its native characteristics and having a negative impact on later protein identification. In each step of conventional polyacrylamide gel assays, information is inevitably lost through protein retention in the gel.